Smith J D, Melián A, Leff T, Breslow J L
Laboratory of Biochemical Genetics and Metabolism, Rockefeller University, New York, New York 10021.
J Biol Chem. 1988 Jun 15;263(17):8300-8.
Apolipoprotein E (apoE), unlike the other major lipoproteins, is synthesized in a variety of tissues. We examined which regions of the human apoE gene contributed to its tissue-specific expression using HepG2 and HeLa cells as examples of expressing and nonexpressing tissues, respectively. Regions between -360 bp and -80 bp and within the first intron were shown to be necessary for full expression activity in HepG2 cells by a nuclease protection assay which demonstrated correct transcriptional initiation of the transfected constructions. To fine map the regulatory regions, we constructed a series of deletions fused to the reporter gene chloramphenicol acetyltransferase. We discovered eight regions which had a positive effect on expression and three regions that had a negative effect on expression, in both HepG2 and HeLa cells. In addition we found three regions which had a tissue-specific negative effect on expression in HeLa cells and one region with a tissue-specific positive effect in HepG2 cells. A DNase I protection assay revealed eight footprints within the proximal 5'-flanking sequence and the first intron. Seven of these footprints fell within closely defined regions with positive expression activity. Sequence analysis of these footprint elements revealed the presence of previously identified elements and two novel elements related to each other, identified here as B1 and B2. We also defined another repeated sequence, the A element; all three of the tissue-nonspecific negative regions contained this element or sequences with homology to it. In the context of a heterologous promotor, a synthetic oligonucleotide containing the B1 and B2 elements behaved like a classical enhancer, having a positive effect on expression, even when placed at a distance. This effect was neutralized by a different synthetic oligonucleotide containing an A element repeat.
与其他主要脂蛋白不同，载脂蛋白E（apoE）在多种组织中合成。我们分别以HepG2细胞和HeLa细胞作为表达组织和非表达组织的示例，研究了人类apoE基因的哪些区域对其组织特异性表达有贡献。通过核酸酶保护试验表明，转染构建体的转录起始正确，结果显示-360 bp至-80 bp之间以及第一个内含子内的区域对于HepG2细胞中的完全表达活性是必需的。为了精细定位调控区域，我们构建了一系列与报告基因氯霉素乙酰转移酶融合的缺失体。我们发现，在HepG2细胞和HeLa细胞中，有八个区域对表达有正向作用，三个区域对表达有负向作用。此外，我们还发现三个区域对HeLa细胞中的表达有组织特异性负向作用，一个区域对HepG2细胞中的表达有组织特异性正向作用。DNase I保护试验揭示了近端5'-侧翼序列和第一个内含子内的八个足迹。这些足迹中的七个位于具有正向表达活性的紧密定义区域内。对这些足迹元件的序列分析揭示了先前鉴定的元件的存在以及两个彼此相关的新元件，在此处鉴定为B1和B2。我们还定义了另一个重复序列，即A元件；所有三个组织非特异性负向区域都包含该元件或与其同源的序列。在异源启动子的背景下，含有B1和B2元件的合成寡核苷酸表现得像一个经典增强子，即使放置在一定距离处也对表达有正向作用。这种作用被含有A元件重复序列的不同合成寡核苷酸所中和。
